Applying symptom dynamics to accurately predict influenza virus infection: An international multicenter influenza‐like illness surveillance study

Abstract Background Public health organizations have recommended various definitions of influenza‐like illnesses under the assumption that the symptoms do not change during influenza virus infection. To explore the relationship between symptoms and influenza over time, we analyzed a dataset from an international multicenter prospective emergency department (ED)‐based influenza‐like illness cohort study. Methods We recruited patients in the US and Taiwan between 2015 and 2020 with: (1) flu‐like symptoms (fever and cough, headache, or sore throat), (2) absence of any of the respiratory infection symptoms, or (3) positive laboratory test results for influenza from the current ED visit. We evaluated the association between the symptoms and influenza virus infection on different days of illness. The association was evaluated among different subgroups, including different study countries, influenza subtypes, and only patients with influenza. Results Among the 2471 recruited patients, 45.7% tested positive for influenza virus. Cough was the most predictive symptom throughout the week (odds ratios [OR]: 7.08–11.15). In general, all symptoms were more predictive during the first 2 days (OR: 1.55–10.28). Upper respiratory symptoms, such as sore throat and productive cough, and general symptoms, such as body ache and fatigue, were more predictive in the first half of the week (OR: 1.51–3.25). Lower respiratory symptoms, such as shortness of breath and wheezing, were more predictive in the second half of the week (OR: 1.52–2.52). Similar trends were observed for most symptoms in the different subgroups. Conclusions The time course is an important factor to be considered when evaluating the symptoms of influenza virus infection.


| INTRODUCTION
Influenza viruses infect approximately one billion people annually, 1 accounting for half of the respiratory infections during the peak of the epidemic. 2 The World Health Organization (WHO) estimates the annual mortality due to influenza to range from 290,000 to 650,000 deaths globally. 1

Timely diagnosis of influenza virus infection is vital
for preventing severe complications. Despite the wide availability of diagnostic tools for influenza virus, clinical gestalt is still needed to increase the pre-test probability and help physicians test for specific pathogens. 3 In the past few decades, many investigators focused on the utility of syndromic surveillance to differentiate influenza from other virus infections. [4][5][6] Unfortunately, there was no single symptom that could be used to detect influenza virus infection with perfect sensitivity. 7,8 To improve the accuracy and to standardize cases for investigation, WHO, the Centers for Disease Control and Prevention (CDC), and the European Centre for Disease Prevention and Control (ECDC) utilized and revised different combinations of symptoms to define influenzalike illness (Table S1). The sensitivities of these definitions, however, are far from perfect, ranging from 32% to 96% (Table S2).
Some researchers have attributed the difficulty in defining influenza-like illness to the dynamic change in symptoms throughout the course. 9,10 Influenza viruses tend to induce a more abrupt onset of symptoms 5,11 than respiratory syncytial virus or rhinoviruses. 12 Whereas patients with influenza tend to have cough and fever simultaneously, patients with rhinovirus usually have cough after nasal symptoms. 13 However, the detailed relationship between symptoms and influenza throughout the course of the disease has not been thoroughly investigated. Previous studies evaluating the association between the symptoms of influenza and the time course were either small virus challenge studies, retrospective patient medical recordbased studies, or studies with a significant amount of missing data. 9,14,15 To explore the symptoms of influenza over time, we analyzed a dataset from an international multicenter prospective emergency department-based influenza-like illness cohort study.

| Study design and setting
Adult patients with fever and flu-like symptoms were recruited in the emergency departments of a surveillance network (Johns Hopkins Centers of Excellence for Influenza Research and Surveillance) in the US and Taiwan between November 2015 and March 2020. This network contained four US hospitals and three hospitals in Taiwan, ranging from tertiary referral medical centers to regional hospitals (Table S3). This study was approved by the Institutional Review Board of Johns Hopkins University (IRB00135664, IRB00041233, IRB00141101, IRB00052743, and IRB00091667) and the Chang Gung Medical Foundation (201406930B0). We followed the Standard for Reporting Diagnostic Accuracy (STARD) for taking history and physical examination to report our manuscript. 16 Adults aged greater than 18 years visited the emergency departments and met any of the inclusion criteria: (1) reported or measured fever and other flu-like symptoms, (2) absence of any of the respiratory infection symptoms, or (3) positive laboratory test results for influenza from the current hospital visit. Flu-like symptoms were defined as any of the three respiratory symptoms: cough, headache, and sore throat. Respiratory infection symptoms were defined as any of fever, cough, headache, sore throat, myalgia (unless due to trauma), rhinorrhea, nasal congestion, or shortness of breath. Eligible patients were interviewed by trained research coordinators using a prespecified and validated data collection form. Patients were excluded if they were unable to provide a written informed consent or had been previously enrolled in the study during the same influenza season.
Patient demographics, comorbidities, history of influenza vaccination, pre-defined symptoms, and days of illness were prospectively investigated by dedicated research coordinators. The first day of illness was defined as the day of symptom onset.

| Laboratory analysis
Influenza virus infection was confirmed by portable polymerase chain reaction (PCR) testing using nasopharyngeal swabs immediately after the interview. The portable PCR-based nucleic acid test was performed using the Xpert Flu Assay, a multiplex PCR platform (Cepheid).
The overall sensitivity and specificity of this platform were both 98% and 100%. 17 In addition, this platform could simultaneously subtype a positive influenza detection.

| Statistical analysis
The differences in numerical means between the influenza-positive and the influenza-negative groups were evaluated using Wilcoxon's rank-sum test or Student's t-test and the categorical variables by the chi-square test. In all investigations, two-sided tests and p-values of 0.05 were considered statistically significant. Odds ratios (OR) were used to measure the strength of the association between symptoms and influenza virus infection. We also measured the proportion of symptoms among influenza-positive patients (sensitivity to detect influenza). To address the relationship between symptoms and influenza at different times, the days of illness were divided into quarters to evaluate the symptom dynamics at presentation. Univariate and multivariate logistic regression analyses were performed to adjust for confounding effects.

| Subgroup and sensitivity analysis
To investigate the potential discrepancy, the analyses were repeated in the following subgroups: history of influenza vaccination, different  Figure 2A).

| Dynamics of predictive symptoms
The patients were further divided into four quartiles according to their days of illness during the first week of symptom onset (Day 1-2, 3, 4-5, and 6-7). More patients with influenza virus infection presented to our emergency departments during the first half of the week after symptom onset (1-3 days: 60.6%; Table S6).
Most of the symptoms were predictive of influenza virus infection during the first 2 days of illness (OR: 1.55-10.28, Figure 2B).

| Subgroup and sensitivity analysis
In the influenza-vaccinated subgroup analysis, the vaccinated patients were older and had more comorbidities, including diabetes, end-stage kidney disease, and asthma, than the unvaccinated patients (Table S7).
In the influenza-vaccinated subgroup, lower respiratory symptoms were predictive of influenza breakthrough infection throughout the week (shortness of breath, OR: 2.06-2.95, Figure 3), and general symptoms, such as headache and chills, were not predictive of influenza virus infection.
In the country-specific subgroup analysis, lower respiratory symptoms were predictive of influenza in the US throughout the week but not of influenza virus infection in the early half of the week in Taiwan ( Figure 4 and Figure S2). In addition, the H3N2 subtypes were more commonly found in the Taiwanese subgroup (Table S8)

| DISCUSSION
In this prospective international multicenter emergency departmentbased cohort study of patients with influenza-like illness, the presentation of influenza was found to vary on different days of illness. Significance tests for the odds ratio were using logistic regression models: *p < 0.05, **p < 0.01, ***p < 0.001.
F I G U R E 3 The odds ratio for symptoms of influenza virus infection with significance in vaccinated subgroup in the days of illness. Upper and lower respiratory symptoms were predictive of influenza throughout the week. General symptoms were not predictive of influenza in first half of the week. Significance tests for the odds ratio using logistic regression models: *p < 0.05, **p < 0.01, ***p < 0.001.

F I G U R E 4
The odds ratio for symptoms of influenza virus infection with significance in the country-specific subgroup analysis in the days of illness. The predictive symptoms in the first half of the week were almost similar in the two countries. Notably, the lower respiratory symptoms were predictive of influenza virus infection in the US throughout the week but not predictive of influenza virus infection in the early half of the week in Taiwan. US, the United States. Significance tests for the odds ratio using logistic regression models: *p < 0.05, **p < 0.01, ***p < 0.001. ∞ Among 51 patients with influenza virus infection in Taiwan on the 6th and 7th day, all patients had the symptom of cough. Therefore, the estimated odds ratio was infinity.  (Table S2). In 2011, the WHO revised the influenzalike illness defined in 1999 to overcome the low accuracy and different phenotypes of the novel H1N1 influenza A virus by omitting the sore throat and adding a time frame. 19 In contrast, the ECDC version of influenza-like illness had relatively high sensitivity but compromised specificity (96.1% vs. 6.6%, Table S2). 20 However, the higher sensitivities of these different versions of influenza-like illness were only found among patients who were enrolled earlier in their illness (Table S2).
Fever and cough were the most common symptoms in the different definitions of influenza-like illness (Table S1). In our study, cough was not only found to be the most predictive symptom (OR: 12.96, 95% CI: 9.28-18.10) but also found to be a consistent predictor of influenza throughout the first week after symptom onset (OR: 7.08 to 11.5). Shah et al also reported that the combination of cough and fever was predictive of influenza virus infection in a retrospective electronic medical record-based study consisting of patients who underwent influenza PCR testing (OR: 6.6). 21 Furthermore, cough was also found to be the most predictive symptom to detect influenza virus infection in previous studies consisting of patients with any flulike symptoms (OR: 6.9 to 13). 4,6 The incidence of cough was higher among influenza patients than among other respiratory viruses, such as rhinovirus, respiratory syncytial virus, and coronavirus. 22 A possible explanation may be the more predominant epithelial cell necrosis that damages airway integrity caused by influenza viruses, compared to other viruses such as rhinovirus and coronavirus. 23 In our study, most of the upper respiratory and general symptoms reported in the first 2 days after symptom onset were more predictive of influenza virus infection than those reported in the latter days.
Some researchers found that the symptom scores of patients with influenza virus and rhinovirus infection peak earlier than those of patients with respiratory syncytial virus infection after symptom onset in prospective studies (influenza and rhinoviruses: 2 days vs. respiratory syncytial virus: 5 days). 11,12 Woolpert et al also found that an abrupt onset of symptom within 3 days was more predictive F I G U R E 5 The odds ratio for symptoms of influenza virus infection with significance in the dominant subtype-specific subgroup analysis in the days of illness. H1N1-and H3N2-dominant season shared the symptoms predictive of influenza virus infection in common. Cough was strongly predictive of influenza virus infection throughout the week. Significance tests for the odds ratio using logistic regression models: *p < 0.05, **p < 0.01, ***p < 0.001.
of influenza virus infection (OR: 3.26). 24 The results could be explained by the higher levels of cytokines, such as interferon-α and 0.41 to 0.72). 5,27 In 2011, the WHO removed sore throat from the influenza-like illness criteria to increase the accuracy after the novel H1N1 influenza outbreak. 19 Our data revealed that sore throat was more predictive of influenza virus infection in the first 3 days. In another prospective study, patients with influenza virus infection experienced sore throat in the earlier days (5.07 days vs. 6.42 days). 28 We believe that the time factor could improve the accuracy of influenza virus infection prediction and could be used to modify influenzalike illness criteria.
In our study, lower respiratory symptoms, such as shortness of breath, wheezing, and chest pain, were associated with influenza virus infection in the second half of the week. These symptoms are more likely to result from complications of influenza virus infection. The most common complication of influenza is pneumonia. 29 The average interval between influenza virus infection and subsequent pneumonia was 6 days after infection, sooner than that for other respiratory viruses (14 days). 30 Contrary to our results, shortness of breath was less likely to be found among patients with influenza virus infection who were recruited only within 3 days after the onset of fever in a prospective cross-sectional study (OR: 0.4). 31 In our subgroup analysis, some predictive symptoms differed between the US and Taiwan, after adjusting for potential confounders. These results could be partially explained by the different distributions of respiratory pathogens between the two countries.
The most common pathogens of respiratory viral infections in the US are rhinovirus, influenza, and respiratory syncytial virus, 32 whereas in Taiwan they are rhinovirus/enterovirus and adenovirus. 33,34 To avoid this spectrum bias, we examined the proportion (or sensitivity) of symptoms only among patients with influenza virus infection. Nevertheless, the sensitivity of symptoms still differed between the two countries after adjusting for possible confounders. A higher proportion of patients with influenza virus infection were found to have lower respiratory symptoms in the US than in Taiwan ( Figure S4). Further studies with larger sample sizes are required to investigate the incidence of lower respiratory symptoms. We further performed dominant subtype-specific subgroup analysis, in which no noticeable differences were found in predictive symptoms between H1N1 and H3N2 strain-dominant seasons. In a systematic review of studies focusing on influenza subtypes, the symptoms of different influenza subtypes were also indistinguishable. 35 Additionally, in our study, general symptoms were not predictive of influenza virus infection in the vaccinated subgroup. Similarly, patients who were vaccinated but still got infected with the influenza virus were significantly more likely to be afebrile with mild symptoms. 36 These breakthrough cases of influenza virus infection were more likely to have shortness of breath than the overall study population throughout the week. We hypothesized that the lower respira-  In conclusion, the time course is an essential factor to be considered when attempting to predict influenza virus infection by symptoms. Cough and rhinorrhea were associated with influenza in the first week. Other upper respiratory and general symptoms were associated with influenza in the first half of the week, whereas lower respiratory symptoms were associated with influenza in the second half of the week. Understanding the time course of influenza symptoms would be helpful for clinicians to treat patients with influenzalike illnesses.

| Protocol
The study protocol is available upon request. The brief description can be found at https://www.niaidceirs.org/resources/ cohort-studies/.